For Western blot analyses, cells were scraped, and lysed in buffer A containing 10 mM HEPES, 10 mM NaCl, 1.5 mM MgCl2, 0.25% Tween 20, 1mM dithiothreitol, 100 mg/ml phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, and 15 mg/ml aprotinin. Thirty μg of total protein from each sample was then loaded onto 10~15% SDS-polyacrylamide gels, because all sorts of proteins measured in this experiment have a diversity of molecular weights (from 4 ~ 120 kDa). Separated proteins were transferred to nitrocellulose membranes (GE Healthcare Life Sciences, Piscataway, NJ, USA), which were blocked with 5% skim milk (at 4°C overnight), and incubated with antibodies against SIRT1 (Santa Cruz, CA), anti-Aβ (6E10) (Covance, Emeryville, CA), and rabbit polyclonal CTFβ (751–770) (Calbiochem, La Jolla CA. Antibodies against beclin-1, Atg5, and LC3 (rabbit monoclonal LC3A/B, 1:1000) were obtained from Cell Signaling Technology (Danvers, MA). Membranes were reprobed with an anti-β-actin antibody (MP Biomedicals, LLC, Aurora, OH, USA) as an internal control.
Anti aβ 6e10
Manufactured by Fortrea
Sourced in United States
The Anti-Aβ (6E10) is a monoclonal antibody that specifically binds to the amyloid-beta (Aβ) protein. It is commonly used in research applications to detect and quantify Aβ in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (3 mentions)
Enhanced chemiluminescence detection kit, by Thermo Fisher Scientific (2 mentions)
Anti bace1, by Merck Group (2 mentions)
Bca protein assay kit, by Beyotime (2 mentions)
Anti actin, by Abcam (2 mentions)
Anti glua1, by Merck Group (2 mentions)
Anti glua2, by Abcam (2 mentions)
Anti phosphorylated glua1 s845, by Merck Group (2 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Antibodies against beclin 1, by Cell Signaling Technology (1 mentions)
Sirt1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin antibody, by MP Biomedicals (1 mentions)
Rifampicin, by Fujifilm (1 mentions)
Anti app c terminal, by Merck Group (1 mentions)
Anti psd 95 antibody, by Abcam (1 mentions)
Bis ans, by Cayman Chemical (1 mentions)
Anti α tubulin, by Merck Group (1 mentions)
Anti iba1, by Fujifilm (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Supersignal west pico, by Thermo Fisher Scientific (1 mentions)
10 protocols using anti aβ 6e10
1
Western Blot Analysis of Protein Markers
2
Antibody Characterization and Reagent Details
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Anti-MITOL rabbit polyclonal antibody was described previously14 (link). Anti-APP C-terminal, anti-α-tubulin, and anti-β-actin antibodies were from Sigma. Anti-Aβ (D54D2), anti-synaptophysin, and anti-presenilin 1 (PS1) antibodies were from Cell Signaling. Anti-normal mouse IgG and anti-Tom20 antibodies were from Santa Cruz Biotechnology. The anti-PSD-95 antibody was from Abcam. Anti-Aβ (6E10) was from Covance. Anti-human Aβ E22P (11A1) antibody was from IBL. A11, OC antibodies were from Thermo Fisher Scientific. Anti-Iba1 was from Wako. Bis-ANS was from Cayman Chemical Company. DAPT and Aβ40 were from PEPTIDE INSTITUTE, INC. Aβ40 was dissolved in 0.02% ammonia solution at 300 µM. To obtain seed-free Aβ40 monomers, these Aβ solutions were centrifuged at 200,000 × g for 3 h at 4 °C and the supernatants were collected. Thus, 0.02% ammonia solution was used as a control in the experiments using either Aβ40. ThS was from Sigma. ThT and Rifampicin were from Wako. Rifampicin was used according to a previous report40 (link). Thus, 0.5% low-viscosity carboxyl methylcellulose (purchased from Sigma) was treated as control.
3
Yeast Protein Extraction and Western Blot
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Yeast cells were harvested and lysed with TCA 85% for 10 min at room temperature (RT). Supernatants were resolved in SDS-PAGE. Gels were transferred in polyvinylidene fluoride membranes (ImmobilonP, Millipore, Burlington, MA, USA), which were blocked for 1 h in tween-tris buffer saline (TTBS) plus 5% milk or 3% bovine serum albumin (BSA). Membranes were incubated o.n. at 4 °C with the following primary antibodies (Abs): 1:500 anti-GFP (Sigma, San Luis, MI, USA) and 1:500 anti-Aβ 6E10 (Covance, Princeton, NJ, USA). Membranes were washed thrice with TTBS and incubated for 1 h with 1:2000 anti-mouse secondary Abs (GE-Healthcare, Chicago, IL, USA). Three washes with TTBS were performed and membranes were developed with Super signal West Pico and Femto Chemiluminiscent substrate (Thermo Scientific, Waltham, MA, USA). Blotting quantification was performed with Quantity One software software (v.4.6.8).
4
Quantifying Amyloid-Beta and BACE1 in Hippocampus
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Frozen hippocampus was homogenized in RIPA buffer supplemented with a protease inhibitor cocktail and centrifuged at 12,000 × g for 20 min at 4°C. The supernatants were collected, and total protein levels were quantified using the BCA protein assay kit (Beyotime, Shanghai, China). A total of 30 μg of each sample was separated by 10% SDS-polyacrylamide gel. After electrophoresis, the proteins were transferred to a nitrocellulose membrane on ice at 100 V for 1 h. The membrane was blocked with 5% w/v skim milk powder in tris buffered saline (TBS-T) containing 0.05% Tween 20 for 1 h. After blocking, the membrane was incubated with a primary antibody overnight at 4°C. The following primary antibodies were used: anti-Aβ (6E10, 1 : 1000, Covance), rabbit polyclonal anti-BACE1 (1 : 1000, Sigma), and anti-β-actin (1 : 5000, Sigma). Immunoreactive bands were detected with HRP-conjugated goat anti-rabbit IgG (1 : 2000, Sigma). The membrane was washed with TBS-T, and the immune complex was visualized using an enhanced chemiluminescence detection kit (Thermo Scientific, USA). The membrane signal was scanned using a FluorChem Scanner and quantified using NIH ImageJ software. These results were normalized to the level of β-actin expression and confirmed by three measurements on the same sample.
5
SDS-PAGE and Western Blot Analysis
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Protein samples for biotinylation and cLTP were loaded on 10% glycine-SDS-PAGE gel. Co-IP samples were loaded on 16% Tricine-SDS-PAGE gel as described (114 (link)). SDS-PAGE gels were transferred to nitrocellulose membranes. The membranes were blocked (5% powdered milk) for 1 h at room temperature, followed by overnight incubation with the primary antibodies at 4 °C. The primary antibodies consisted of anti-GluA1 (Millipore, 1:2000), anti-GluA2 (Abcam, 1:2000), anti-phosphorylated GluA1 S845 (Millipore, 1:1000), anti-GFP (Torrey Pines, 1:2000), anti-Aβ (6E10, Covance, 1:2000), and anti-actin (Abcam, 1:2000) antibodies. Membranes were subsequently incubated by secondary antibodies for 1 h at room temperature and developed with Enhanced Chemiluminescence (Thermo Fisher Scientific). Protein bands were quantified using ImageJ (https://imagej.nih.gov/ij/ ). Immunoblots were at least duplicated for quantitative analysis.
6
Hippocampal Protein Extraction and Analysis
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Protein extraction from the hippocampus was performed as previously described [19 (link)]. Protein concentration was determined using the BCA protein assay (Tiangen Biotech Co. Ltd., Beijing, China). 10 µg of protein from each sample was separated by 10% SDS-PAGE and electrotransferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) for immunoblotting. The following primary antibodies were used: anti-Aβ (6E10, 1:1000; Covance, Emeryville, CA, USA), anti-BACE1 (1:500, ab2077; Abcam, Cambridge, MA, USA), and anti-β-Actin (1:500, ab2077; Abcam). Band intensities were semi-quantified using ImageJ ver. 1.6 (http://imagej.nih.gov/ij/ ) software.
7
Western Blot Analysis of Synaptic Proteins
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Protein samples for biotinylation and cLTP were loaded on 10% glycine-SDS-PAGE gel. Co-IP samples were loaded 16% Tricine-SDS-PAGE gel as described previously (89) . SDS-PAGE gels were transferred to nitrocellulose membranes. The membranes were blocked (5% powdered milk) for 1 h at room temperature, followed by overnight incubation with the primary antibodies at 4°C. The primary antibodies consisted of anti-GluA1 (Millipore, 1:2000), anti-GluA2 (Abcam, 1:2000), antiphosphorylated GluA1 S845 (Millipore, 1:1000), anti-GFP (Torrey Pines, 1:2000), anti-Aβ (6E10, Covance, 1:2000), and anti-actin (Abcam, 1:2000) antibodies. Membranes were subsequently incubated by secondary antibodies for 1 h at room temperature and developed with Enhanced Chemiluminescence (ECL) (Thermo Fisher Scientific, Waltham, MA). Protein bands were quantified using ImageJ (https://imagej.nih.gov/ij/). Immunoblots were at least duplicated for quantitative analysis.
8
Quantifying Amyloid-Beta in Brain Samples
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Immunoassay.Microplates Microlon/F-shape REF 655092 (Greiner) were coated with 1 µg/mL of human 3D6 (28)overnight at room temperature in coating buffer (sodium carbonate pH=9.6 0.05M NaCO 3 in MQ water). After washing plates (washing buffer 0.05% Tween-20 in PBS were blocked with 4% not fat dry milk and incubated with brain homogenates or with Aβto generate the standard curve. Next plates were washed, incubated with 50ng/mL of biotinylated human 20C2(28) and washed again. Finally, plates were incubated with streptavidin-HRP (Jackson ImmunoResearch Laboratories) diluted 1:8,000 and developed using 3,3',5,5'-Tetramethylbenzidine (TMB).The absorption was measured at 450 nm within 30 minutes of stopping the reaction with 2 M H 2 SO 4 using the Perkin Elmer 2030 manager system. Western blot. TBS and TBS-T samples corresponding to 40 µg of total protein we loaded onto a precast TGX 4-16% gel (Bio-Rad). Then samples were transferred to PDVF membranes, blocked with Odyssey blocking buffer and probed with 1µg mAb anti-Aβ (6E10, Covance). After incubation with donkey antimouse IRdye680 (Rockland Immunochemicals) diluted 1:1,000 in Odyssey blocking buffer, the membrane was scanned and analyzed with Odyssey imager Li-Cor. The intensities of the APP bands detected at 100 kDa, were measured with Odyssey imager Li-Cor.
9
Western Blot Analysis of Alzheimer's Biomarkers
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Frozen hippocampi were homogenized in RIPA buffer supplemented with protease inhibitor cocktail and centrifuged at 12,000 × g for 20 min at 4°C. The supernatants were collected, and total protein levels were quantified using a BCA protein assay kit (Beyotime, Haimen, Jiangsu, China). Equal amounts (30 μg) of each sample were separated on 10% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred onto a nitrocellulose membrane (Millipore) at 100 V for 60 min on ice. The membrane was blocked with 5% w/v non-fat dry milk powder in Tris-buffered saline with 0.05% Tween 20 (TBS-T) for 1 h. After blocking, membranes were incubated with the primary antibodies overnight at 4°C. The following primary antibodies were used: anti-Aβ (6E10, 1:1000, Covance), rabbit polyclonal anti-BACE1 (1:1000, Sigma), and anti-β-actin (1:5000, Sigma). Immunoreactive bands were detected with HRP-conjugated goat anti-rabbit IgG (1:2000, Santa Cruz Biotechnology). The membrane was washed with TBS-T and the immunocomplex was visualized using an enhanced chemiluminescence detection kit (Thermo Scientific, Rockford, IL, USA). Signals of the membrane were scanned using the FluorChem Scanner and quantified using NIH Image J software. These results were normalized against β-actin expression levels and confirmed by triplicate measurements of the same sample.
10
Immunostaining of Mouse Hemibrains
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